Expression of the plastocyanin gene PETE2 in Camelina sativa improves seed yield and salt tolerance.

Plastocyanin functions as an electron carrier in the photosynthetic electron transport chain, located at the thylakoid membrane. In several species, endogenous plastocyanin levels are correlated with the photosynthetic electron transport rate. Overexpression of plastocyanin genes in Arabidopsis thaliana increases plant size, but this phenomenon has not been observed in crop species. Here, we investigated the effects of heterologous expression of a gene encoding a plastocyanin isoform from Arabidopsis, AtPETE2, in the oil seed crop Camelina sativa under standard growth conditions and under salt stress. AtPETE2 heterologous expression enhanced photosynthetic activity in Camelina, accelerating plant development and improving seed yield under standard growth conditions. Additionally, CsPETE2 from Camelina was induced by salt stress and AtPETE2 expression lines had larger primary roots and more lateral roots than the wild type. AtPETE2 expression lines also had larger seeds and higher total seed yield under long-term salt stress compared with non-transgenic Camelina. Our results demonstrate that increased plastocyanin levels in Camelina can enhance photosynthesis and productivity, as well as tolerance to osmotic and salt stresses. Heterologous expression of plastocyanin may be a useful strategy to mitigate crop stress in saline soils.

Loss of a pyridoxal-phosphate phosphatase rescues Arabidopsis lacking an endoplasmic reticulum ATP carrier.

The endoplasmic reticulum (ER)-located ATP/ADP-antiporter (ER-ANT1) occurs specifically in vascular plants. Structurally different transporters mediate energy provision to the ER, but the cellular function of ER-ANT1 is still unknown. Arabidopsis (Arabidopsis thaliana) mutants lacking ER-ANT1 (er-ant1 plants) exhibit a photorespiratory phenotype accompanied by high glycine levels and stunted growth, pointing to an inhibition of glycine decarboxylase (GDC). To reveal whether it is possible to suppress this marked phenotype, we exploited the power of a forward genetic screen. Absence of a so far uncharacterized member of the HaloAcid Dehalogenase (HAD)-like hydrolase family strongly suppressed the dwarf phenotype of er-ant1 plants. Localization studies suggested that the corresponding protein locates to chloroplasts, and activity assays showed that the enzyme dephosphorylates, with high substrate affinity, the B6 vitamer pyridoxal 5'-phosphate (PLP). Additional physiological experiments identified imbalances in vitamin B6 homeostasis in er-ant1 mutants. Our data suggest that impaired chloroplast metabolism, but not decreased GDC activity, causes the er-ant1 mutant dwarf phenotype. We present a hypothesis, setting transport of PLP by ER-ANT1 and chloroplastic PLP dephosphorylation in the cellular context. With the identification of this HAD-type PLP phosphatase, we also provide insight into B6 vitamer homeostasis.

Overexpression of the vacuolar sugar importer BvTST1 from sugar beet in Camelina improves seed properties and leads to altered root characteristics.

Overexpression of the vacuolar sugar transporter TST1 in Arabidopsis leads to higher seed lipid levels and higher total seed yield per plant. However, effects on fruit biomass have not been observed in crop plants like melon, strawberry, cotton, apple, or tomato with increased tonoplast sugar transporter (TST) activity. Thus, it was unclear whether overexpression of TST in selected crops might lead to increased fruit yield, as observed in Arabidopsis. Here, we report that constitutive overexpression of TST1 from sugar beet in the important crop species Camelina sativa (false flax) resembles the seed characteristics observed for Arabidopsis upon increased TST activity. These effects go along with a stimulation of sugar export from source leaves and not only provoke optimised seed properties like higher lipid levels and increased overall seed yield per plant, but also modify the root architecture of BvTST1 overexpressing Camelina lines. Such mutants grew longer primary roots and showed an increased number of lateral roots, especially when developed under conditions of limited water supply. These changes in root properties result in a stabilisation of total seed yield under drought conditions. In summary, we demonstrate that increased vacuolar TST activity may lead to optimised yield of an oil-seed crop species with high levels of healthy ω3 fatty acids in storage lipids. Moreover, since BvTST1 overexpressing Camelina mutants, in addition, exhibit optimised yield under limited water availability, we might devise a strategy to create crops with improved tolerance against drought, representing one of the most challenging environmental cues today and in future.

Ectopic maltase alleviates dwarf phenotype and improves plant frost tolerance of maltose transporter mutants.

Maltose, the major product of starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves, exits the chloroplast via the maltose exporter1 MEX1. Consequently, mex1 loss-of-function plants exhibit substantial maltose accumulation, a starch-excess phenotype and a specific chlorotic phenotype during leaf development. Here, we investigated whether the introduction of an alternative metabolic route could suppress the marked developmental defects typical for mex1 loss-of-function mutants. To this end, we ectopically expressed in mex1  chloroplasts a functional maltase (MAL) from baker's yeast (Saccharomyces cerevisiae, chloroplastidial MAL [cpMAL] mutants). Remarkably, the stromal MAL activity substantially alleviates most phenotypic peculiarities typical for mex1 plants. However, the cpMAL lines contained only slightly less maltose than parental mex1 plants and their starch levels were, surprisingly, even higher. These findings point to a threshold level of maltose responsible for the marked developmental defects in mex1. While growth and flowering time were only slightly retarded, cpMAL lines exhibited a substantially improved frost tolerance, when compared to wild-types. In summary, these results demonstrate the possibility to bypass the MEX1 transporter, allow us to differentiate between possible starch-excess and maltose-excess responses, and demonstrate that stromal maltose accumulation prevents frost defects. The latter insight may be instrumental for the development of crop plants with improved frost tolerance.

The Tapetal Major Facilitator NPF2.8 Is Required for Accumulation of Flavonol Glycosides on the Pollen Surface in Arabidopsis thaliana.

The exine of angiosperm pollen grains is usually covered by a complex mix of metabolites including pollen-specific hydroxycinnamic acid amides (HCAAs) and flavonoid glycosides. Although the biosynthetic pathways resulting in the formation of HCAAs and flavonol glycosides have been characterized, it is unclear how these compounds are transported to the pollen surface. In this report we provide several lines of evidence that a member of the nitrate/peptide transporter family is required for the accumulation and transport of pollen-specific flavonol 3-o-sophorosides, characterized by a glycosidic ß-1,2-linkage, to the pollen surface of Arabidopsis (Arabidopsis thaliana). Ectopic, transient expression in Nicotiana benthamiana epidermal leaf cells demonstrated localization of this flavonol sophoroside transporter (FST1) at the plasmalemma when fused to green fluorescent protein (GFP). We also confirmed the tapetum-specific expression of FST1 by GFP reporter lines driven by the FST1 promoter. In vitro characterization of FST1 activity was achieved by microbial uptake assays based on 14C-labeled flavonol glycosides. Finally, rescue of an fst1 insertion mutant by complementation with an FST1 genomic fragment restored the accumulation of flavonol glycosides in pollen grains to wild-type levels, corroborating the requirement of FST1 for transport of flavonol-3-o-sophorosides from the tapetum to the pollen surface.

Identification of Chloroplast Envelope Proteins with Critical Importance for Cold Acclimation.

The ability of plants to withstand cold temperatures relies on their photosynthetic activity. Thus, the chloroplast is of utmost importance for cold acclimation and acquisition of freezing tolerance. During cold acclimation, the properties of the chloroplast change markedly. To provide the most comprehensive view of the protein repertoire of the chloroplast envelope, we analyzed this membrane system in Arabidopsis (Arabidopsis thaliana) using mass spectrometry-based proteomics. Profiling chloroplast envelope membranes was achieved by a cross comparison of protein intensities across the plastid and the enriched membrane fraction under both normal and cold conditions. We used multivariable logistic regression to model the probabilities for the classification of an envelope localization. In total, we identified 38 envelope membrane intrinsic or associated proteins exhibiting altered abundance after cold acclimation. These proteins comprise several solute carriers, such as the ATP/ADP antiporter nucleotide transporter2 (NTT2; substantially increased abundance) or the maltose exporter MEX1 (substantially decreased abundance). Remarkably, analysis of the frost recovery of ntt loss-of-function and mex1 overexpressor mutants confirmed that the comparative proteome is well suited to identify key factors involved in cold acclimation and acquisition of freezing tolerance. Moreover, for proteins with known physiological function, we propose scenarios explaining their possible roles in cold acclimation. Furthermore, spatial proteomics introduces an additional layer of complexity and enables the identification of proteins differentially localized at the envelope membrane under the changing environmental regime.

Nucleotide Transport and Metabolism in Diatoms.

Plastids, organelles that evolved from cyanobacteria via endosymbiosis in eukaryotes, provide carbohydrates for the formation of biomass and for mitochondrial energy production to the cell. They generate their own energy in the form of the nucleotide adenosine triphosphate (ATP). However, plastids of non-photosynthetic tissues, or during the dark, depend on external supply of ATP. A dedicated antiporter that exchanges ATP against adenosine diphosphate (ADP) plus inorganic phosphate (Pi) takes over this function in most photosynthetic eukaryotes. Additional forms of such nucleotide transporters (NTTs), with deviating activities, are found in intracellular bacteria, and, surprisingly, also in diatoms, a group of algae that acquired their plastids from other eukaryotes via one (or even several) additional endosymbioses compared to algae with primary plastids and higher plants. In this review, we summarize what is known about the nucleotide synthesis and transport pathways in diatom cells, and discuss the evolutionary implications of the presence of the additional NTTs in diatoms, as well as their applications in biotechnology.

The Plastidic Sugar Transporter pSuT Influences Flowering and Affects Cold Responses.

Sucrose (Suc) is one of the most important types of sugars in plants, serving inter alia as a long-distance transport molecule, a carbon and energy storage compound, an osmotically active solute, and fuel for many anabolic reactions. Suc biosynthesis and degradation pathways are well known; however, the regulation of Suc intracellular distribution is poorly understood. In particular, the cellular function of chloroplast Suc reserves and the transporters involved in accumulating these substantial Suc levels remain uncharacterized. Here, we characterize the plastidic sugar transporter (pSuT) in Arabidopsis (Arabidopsis thaliana), which belongs to a subfamily of the monosaccharide transporter-like family. Transport analyses with yeast cells expressing a truncated, vacuole-targeted version of pSuT indicate that both glucose and Suc act as substrates, and nonaqueous fractionation supports a role for pSuT in Suc export from the chloroplast. The latter process is required for a correct transition from vegetative to reproductive growth and influences inflorescence architecture. Moreover, pSuT activity affects freezing-induced electrolyte release. These data further underline the central function of the chloroplast for plant development and the modulation of stress tolerance.

PAPST2 Plays Critical Roles in Removing the Stress Signaling Molecule 3'-Phosphoadenosine 5'-Phosphate from the Cytosol and Its Subsequent Degradation in Plastids and Mitochondria.

The compartmentalization of PAPS (the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate) synthesis (mainly in plastids), PAPS consumption (in the cytosol), and PAP (the stress signaling molecule 3'-phosphoadenosine 5'-phosphate) degradation (in plastids and mitochondria) requires organellar transport systems for both PAPS and PAP. The plastidial transporter PAPST1 (PAPS TRANSPORTER1) delivers newly synthesized PAPS from the stroma to the cytosol. We investigated the activity of PAPST2, the closest homolog of PAPST1, which unlike PAPST1 is targeted to both the plastids and mitochondria. Biochemical characterization in Arabidopsis thaliana revealed that PAPST2 mediates the antiport of PAP, PAPS, ATP, and ADP. Strongly increased cellular PAP levels negatively affect plant growth, as observed in the fry1 papst2 mutant, which lacks the PAP-catabolizing enzyme SALT TOLERANCE 1 and PAPST2. PAP levels were specifically elevated in the cytosol of papst2 and fiery1 papst2, but not in papst1 or fry1 papst1 PAPST1 failed to complement the papst2 mutant phenotype in mitochondria, because it likely removes PAPS from the cell, as demonstrated by the increased expression of phytosulfokine genes. Overexpression of SAL1 in mitochondria rescued the phenotype of fry1 but not fry1 papst2 Therefore, PAPST2 represents an important organellar importer of PAP, providing a piece of the puzzle in our understanding of the organelle-to-nucleus PAP retrograde signaling pathway.

AXER is an ATP/ADP exchanger in the membrane of the endoplasmic reticulum.

To fulfill its role in protein biogenesis, the endoplasmic reticulum (ER) depends on the Hsp70-type molecular chaperone BiP, which requires a constant ATP supply. However, the carrier that catalyzes ATP uptake into the ER was unknown. Here, we report that our screen of gene expression datasets for member(s) of the family of solute carriers that are co-expressed with BiP and are ER membrane proteins identifies SLC35B1 as a potential candidate. Heterologous expression of SLC35B1 in E. coli reveals that SLC35B1 is highly specific for ATP and ADP and acts in antiport mode. Moreover, depletion of SLC35B1 from HeLa cells reduces ER ATP levels and, as a consequence, BiP activity. Thus, human SLC35B1 may provide ATP to the ER and was named AXER (ATP/ADP exchanger in the ER membrane). Furthermore, we propose an ER to cytosol low energy response regulatory axis (termed lowER) that appears as central for maintaining ER ATP supply.

Reduced vacuolar ß-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum.

The ß-1,3-glucan chrysolaminarin is the main storage polysaccharide of diatoms. In contrast to plants and green algae, diatoms and most other algal groups do not accumulate storage polysaccharides in their plastids. The diatom Phaeodactylum tricornutum possesses only a single gene encoding a putative ß-1,3-glucan synthase (PtBGS). Here, we characterize this enzyme by expressing GFP fusion proteins in P. tricornutum and by creating and investigating corresponding gene silencing mutants. We demonstrate that PtBGS is a vacuolar protein located in the tonoplast. Metabolite analyses of two mutant strains with reduced amounts of PtBGS reveal a reduction in their chrysolaminarin content and an increase of soluble sugars and lipids. This indicates that carbohydrates are shunted into alternative pathways when chrysolaminarin production is impaired. The mutant strains show reduced growth and lower photosynthetic capacities, while possessing higher photoprotective abilities than WT cells. Interestingly, a strong reduction in PtBGS expression also results in aberrations of the usually very regular thylakoid membrane patterns, including increased thylakoid thickness, reduced numbers of thylakoids per plastid, and increased numbers of lamellae per thylakoid stack. Our data demonstrate the complex intertwinement of carbohydrate storage in the vacuoles with carbohydrate metabolism, photosynthetic homeostasis, and plastid morphology.

Crossing the border - Solute entry into the chlamydial inclusion.

Chlamydiales comprise important human and animal pathogens as well as endosymbionts of amoebae. Generally, these obligate intracellular living bacteria are characterized by a biphasic developmental cycle, a reduced genome and a restricted metabolic capacity. Because of their metabolic impairment, Chlamydiales essentially rely on the uptake of diverse metabolites from their hosts. Chlamydiales thrive in a special compartment, the inclusion, and hence are surrounded by an additional membrane. Solutes might enter the inclusion through pores and open channels or by redirection of host vesicles, which fuse with the inclusion membrane and release their internal cargo. Recent investigations shed new light on the chlamydia-host interaction and identified an additional way for nutrient uptake into the inclusion. Proteome studies and targeting analyses identified chlamydial and host solute carriers in inclusions of Chlamydia trachomatis infected cells. These transporters are involved in the provision of UDP-glucose and biotin, and probably deliver further metabolites to the inclusion. By the controlled recruitment of specific solute carriers to the inclusion, the chlamydial resident thus can actively manipulate the metabolite availability and composition in the inclusion. This review summarizes recent findings and new ideas on carrier mediated solute uptake into the chlamydial inclusion in the context of the bacterial and host metabolism.

Shuttling of (deoxy-) purine nucleotides between compartments of the diatom Phaeodactylum tricornutum.

Diatom plastids show several peculiarities when compared with primary plastids of higher plants or algae. They are surrounded by four membranes and depend on nucleotide uptake because, unlike in plants, nucleotide de novo synthesis exclusively occurs in the cytosol. Previous analyses suggest that two specifically adapted nucleotide transporters (NTTs) facilitate the required passage of nucleotides across the innermost plastid membrane. However, nucleotide transport across the additional plastid membranes remains to be clarified. Phylogenetic studies, transport assays with the recombinant protein as well as GFP-based targeting analyses allowed detailed characterization of a novel isoform (PtNTT5) of the six NTTs of Phaeodactylum tricornutum. PtNTT5 exhibits low amino acid similarities and is only distantly related to all previously characterized NTTs. However, in a heterologous expression system, it acts as a nucleotide antiporter and prefers various (deoxy-) purine nucleotides as substrates. Interestingly, PtNTT5 is probably located in the endoplasmic reticulum, which in diatoms also represents the outermost plastid membrane. PtNTT5, with its unusual transport properties, phylogeny and localization, can be taken as further evidence for the establishment of a sophisticated and specifically adapted nucleotide transport system in diatom plastids.

In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca(2+)-effects.

BACKGROUND: Adenine nucleotide/phosphate carriers (APCs) from mammals and yeast are commonly known to adapt the mitochondrial adenine nucleotide pool in accordance to cellular demands. They catalyze adenine nucleotide--particularly ATP-Mg--and phosphate exchange and their activity is regulated by calcium. Our current knowledge about corresponding proteins from plants is comparably limited. Recently, the three putative APCs from Arabidopsis thaliana were shown to restore the specific growth phenotype of APC yeast loss-of-function mutants and to interact with calcium via their N-terminal EF--hand motifs in vitro. In this study, we performed biochemical characterization of all three APC isoforms from A. thaliana to gain further insights into their functional properties. RESULTS: Recombinant plant APCs were functionally reconstituted into liposomes and their biochemical characteristics were determined by transport measurements using radiolabeled substrates. All three plant APCs were capable of ATP, ADP and phosphate exchange, however, high preference for ATP-Mg, as shown for orthologous carriers, was not detectable. By contrast, the obtained data suggest that in the liposomal system the plant APCs rather favor ATP-Ca as substrate. Moreover, investigation of a representative mutant APC protein revealed that the observed calcium effects on ATP transport did not primarily/essentially involve Ca(2+)-binding to the EF-hand motifs in the N-terminal domain of the carrier. CONCLUSION: Biochemical characteristics suggest that plant APCs can mediate net transport of adenine nucleotides and hence, like their pendants from animals and yeast, might be involved in the alteration of the mitochondrial adenine nucleotide pool. Although, ATP-Ca was identified as an apparent import substrate of plant APCs in vitro it is arguable whether ATP-Ca formation and thus the corresponding transport can take place in vivo.

Habitat stress initiates changes in composition, CO2 gas exchange and C-allocation as life traits in biological soil crusts.

Biological soil crusts (BSC) are the dominant functional vegetation unit in some of the harshest habitats in the world. We assessed BSC response to stress through changes in biotic composition, CO2 gas exchange and carbon allocation in three lichen-dominated BSC from habitats with different stress levels, two more extreme sites in Antarctica and one moderate site in Germany. Maximal net photosynthesis (NP) was identical, whereas the water content to achieve maximal NP was substantially lower in the Antarctic sites, this apparently being achieved by changes in biomass allocation. Optimal NP temperatures reflected local climate. The Antarctic BSC allocated fixed carbon (tracked using (14)CO2) mostly to the alcohol soluble pool (low-molecular weight sugars, sugar alcohols), which has an important role in desiccation and freezing resistance and antioxidant protection. In contrast, BSC at the moderate site showed greater carbon allocation into the polysaccharide pool, indicating a tendency towards growth. The results indicate that the BSC of the more stressed Antarctic sites emphasise survival rather than growth. Changes in BSC are adaptive and at multiple levels and we identify benefits and risks attached to changing life traits, as well as describing the ecophysiological mechanisms that underlie them.

From endoplasmic reticulum to mitochondria: absence of the Arabidopsis ATP antiporter endoplasmic Reticulum Adenylate Transporter1 perturbs photorespiration.

The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO(2) concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.

The endosymbiont Amoebophilus asiaticus encodes an S-adenosylmethionine carrier that compensates for its missing methylation cycle.

All organisms require S-adenosylmethionine (SAM) as a methyl group donor and cofactor for various biologically important processes. However, certain obligate intracellular parasitic bacteria and also the amoeba symbiont Amoebophilus asiaticus have lost the capacity to synthesize this cofactor and hence rely on its uptake from host cells. Genome analyses revealed that A. asiaticus encodes a putative SAM transporter. The corresponding protein was functionally characterized in Escherichia coli: import studies demonstrated that it is specific for SAM and S-adenosylhomocysteine (SAH), the end product of methylation. SAM transport activity was shown to be highly dependent on the presence of a membrane potential, and by targeted analyses, we obtained direct evidence for a proton-driven SAM/SAH antiport mechanism. Sequence analyses suggest that SAM carriers from Rickettsiales might operate in a similar way, in contrast to chlamydial SAM transporters. SAM/SAH antiport is of high physiological importance, as it allows for compensation for the missing methylation cycle. The identification of a SAM transporter in A. asiaticus belonging to the Bacteroidetes phylum demonstrates that SAM transport is more widely spread than previously assumed and occurs in bacteria belonging to three different phyla (Proteobacteria, Chlamydiae, and Bacteroidetes).

Current progress in tonoplast proteomics reveals insights into the function of the large central vacuole.

Vacuoles of plants fulfill various biologically important functions, like turgor generation and maintenance, detoxification, solute sequestration, or protein storage. Different types of plant vacuoles (lytic versus protein storage) are characterized by different functional properties apparently caused by a different composition/abundance and regulation of transport proteins in the surrounding membrane, the tonoplast. Proteome analyses allow the identification of vacuolar proteins and provide an informative basis for assigning observed transport processes to specific carriers or channels. This review summarizes techniques required for vacuolar proteome analyses, like e.g., isolation of the large central vacuole or tonoplast membrane purification. Moreover, an overview about diverse published vacuolar proteome studies is provided. It becomes evident that qualitative proteomes from different plant species represent just the tip of the iceberg. During the past few years, mass spectrometry achieved immense improvement concerning its accuracy, sensitivity, and application. As a consequence, modern tonoplast proteome approaches are suited for detecting alterations in membrane protein abundance in response to changing environmental/physiological conditions and help to clarify the regulation of tonoplast transport processes.

The Arabidopsis thylakoid ADP/ATP carrier TAAC has an additional role in supplying plastidic phosphoadenosine 5'-phosphosulfate to the cytosol.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the high-energy sulfate donor for sulfation reactions. Plants produce some PAPS in the cytosol, but it is predominantly produced in plastids. Accordingly, PAPS has to be provided by plastids to serve as a substrate for sulfotransferase reactions in the cytosol and the Golgi apparatus. We present several lines of evidence that the recently described Arabidopsis thaliana thylakoid ADP/ATP carrier TAAC transports PAPS across the plastid envelope and thus fulfills an additional function of high physiological relevance. Transport studies using the recombinant protein revealed that it favors PAPS, 3'-phosphoadenosine 5'-phosphate, and ATP as substrates; thus, we named it PAPST1. The protein could be detected both in the plastid envelope membrane and in thylakoids, and it is present in plastids of autotrophic and heterotrophic tissues. TAAC/PAPST1 belongs to the mitochondrial carrier family in contrast with the known animal PAPS transporters, which are members of the nucleotide-sugar transporter family. The expression of the PAPST1 gene is regulated by the same MYB transcription factors also regulating the biosynthesis of sulfated secondary metabolites, glucosinolates. Molecular and physiological analyses of papst1 mutant plants indicate that PAPST1 is involved in several aspects of sulfur metabolism, including the biosynthesis of thiols, glucosinolates, and phytosulfokines.

The membrane insertase Oxa1 is required for efficient import of carrier proteins into mitochondria.

Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady-state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperature-sensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin.